Ultracentrifuge containers or sample tubes have been developed in a variety of sizes, materials, wall thicknesses and sealing means. Tube materials provide a range of chemical resistance, operating conditions, and clarity for observing samples. Wall thickness provides a range of operating speeds, and sample retrieval by puncturing or slicing of the tube. Sealing means include those of heat sealing, screw caps with elastomeric gaskets, crimping, swaging and compressing all to provide degrees of reliability and reusability of the tubes. A range of diameters and lengths provide convenient chambers for particular separations within time and speed constraints. Until recently, all ultracentrifuge tubes were manufactured with cylindrical side walls closed on the bottom with a hemisphere.
Ultracentrifuge tubes with hemispherical bottoms have proven to be the most easily removed from ultracentrifuge rotor holes. Lower speed centrifuge applications, typically less than 25,000 rpm, do not generate sufficient hydrostatic pressures to deleteriously affect the removal of tubes from rotor holes. Ultracentrifuge rotors spinning in the range of about 25,000 to about 100,000 rpm generate extremely high pressures in the liquid contents of the tubes and cause creep, or time-dependent deformation of the tube dimensions. Particularly at the bottom portions of the tube forces of 600,000 g's may be encountered. Pressures of 32,000 psi can exist dependent on the rotor speed and the sample mass. Thus a sample tube weighing just 3 grams may well have a weight of 1 ton in use due to the centrifugal forces. After centrifugation has been completed, appreciable time is required to allow relaxation and removal of the tubes from the rotors. It has been found that a rotor hole with a cylindrical top section and a hemispherical bottom and corresponding to the tube shape facilitates the removal of the tube, as the mating surfaces of the rotor and tube hemispheres provide the least possibility of taper or locking interference.
One disadvantage of the cylindrical tube with a hemispherical bottom is the relatively large surface area provided by the bottom of the tube with the result that sample material to be collected, i.e., a band of precipitate or other desire fraction, is dispersed over the hemispherical internal surface of the tube. An improved tube configuration for sample concentration is a tube with a cylindrical upper section closed on the bottom with a reduced diameter hemisphere, the transition between diameters being accomplished through a conical section. This tube is commonly referred to as a conical tube.
For many years the conical tube has been used in low, medium and so-called superspeed centrifuges up to the rotating speeds of approximately 25,000 rpm. The conical tubes in these applications are made with thick walls, e.g., 0.050 inches (1.5 mm) thick, and are self-supporting in that liquid support by the tube contents is not required to prevent collapse of the tube. Adapters are provided which support the tube in the conical transition between the cylindrical upper portion and the reduced diameter hemispherical portion. The adapters are often made of rigid polymer materials such as polyacetal and polypropylene or elastomers such as Neoprene and thermoplastic rubbers. In these cases the pressures generated by the liquid inside the tube are not high, and the adapters are normally easily extracted from the rotor hole and the tubes easily extracted from the adapters.
Lam and Williams, "Multiangle Adapter for Fixed Angle Centrifuge Rotor," U.S. Pat. No. 4,553,955, describes a centrifuge rotor into which is inserted a long-length adapter at selected rotational alignment to provide various angles of use of centrifuge tubes with a conical transition.
Anthony, "Split Tube Centrifuge Rotor Adapter," U.S. Pat. No. 4,692,137, describes a long-length centrifuge adapter split in two sections longitudinally to facilitate the removal of straight-wall tubes with hemispherical bottoms from the adapters.
Romanauskas, "Inside Adapter for a Sample Container," U.S Pat. No. 4,451,250 describes an application in vertical rotors where the rotor spin axis is coincident with the longitudinal axis of the tube. An adapter is placed inside the normally sealed centrifuge tube and reduced volume of fluid sample resides in a sector-shaped chamber in the adapter.
Adapters for open-top tubes in swinging bucket and fixed angle rotors have previously been developed. Typically the adapters are constructed of polyacetal, a strong, lubricious and dense thermoplastic material. The adapters are sold by many centrifuge companies. The adapters extend to the top of the rotor hole, and a sample tube of a lessor diameter than the rotor hole fits inside the adapter. The speed of centrifugation must be reduced in consideration of the adapter density and the drop of liquid level caused by the additional clearance of the tube diameter in the adapter. Because of reduced speed and extension of the adapter to the top of the rotor hole, removal of the tube from the adapter and the adapter from the hole is relatively easily accomplished.
One recent application of conical tubes in fixed angle rotors is the use of a microcentrifuge tube with attached cap in conjunction with a plastic adapter. The combination of such tube and adapter is sold by Beckman Instruments, Inc. for use in their product number TLA-100.3, fixed angle rotor, and is described as a "conical bottom microcentrifuge tube with special adapters." The microcentrifuge tube is a rigid, self-supporting container with a cap and must be used at speeds reduced 50% from the maximum operating speed of the rotor which is 100,000 rpm. The tube and adapter fit into a hole which is inclined 30.degree. from the spin axis of the rotor. The outside diameter of the overall adapter is straight and slightly less than the inside diameter of the rotor hole. The rotor speed reduction is presumably required to prevent leakage of the tube cap and to facilitate removal of the adapter which otherwise tends to become fixed in the rotor hole.
The first application of a thin wall, open top, conical tube in an ultracentrifuge rotor is sold by Seton Scientific Co. under the trademark "UltraCone." The primary application is in swinging bucket rotors where the longitudinal axis of the tube and rotor during ultracentrifuging is at 90.degree. to the spin axis of the rotor. Thin wall tubes are used for achieving maximum fluid volume and for ease of puncturing the tube wall for sample retrieval at various levels of a tube subsequently removed from the rotor. The conical tube/adapter combination can be used at the maximum speed and density of which the rotor is capable. The adapter is made of a rigid polymer material such a Noryl plastic, and the configuration of the adapter outside surface is a cylinder adjacent to a hemispherical bottom section. The adapter internal surface is a tapered surface adjacent to a hemispherical bottom, and the conical portion of the tube placed into the adapter conforms exactly to the internal mating surface provided by the adapter. The adapter is used at the bottom of the rotor hole under the conical region of the tube, and the upper cylindrical portion of the tube fits the rotor hole. The thin wall tube must be completely filled with fluid sample in order to provide adequate support to the tube wall during centrifugation.
One problem encountered in the use of the Seton UltraCone adapter is the tendency for the adapter to become fixed (stuck) in the bottom of the rotor bucket particularly after long runs at high temperatures. Various thin wall thermoplastic conical tubes when used with an adapter made of a rigid material such as a modified phenylene-oxide based resin also become fixed in the adapter. Both circumstances cause difficulty to the centrifuge user because the separated components of the tube original sample components can be agitated and remixed during the attempt to remove a sticking tube/adapter from the rotor hole. Another problem encountered is the cracking of the adapters after repeated use. The amount of strain experienced by the adapter in tension as it expands to meet the also expanding rotor bucket is not considered high when compared to noncentrifugal strain levels in other plastic applications, but the stress level is extremely high and causes low cycle fatigue in a short period of time. The effects of fatigue are increased by temperature. In ultracentrifugation 20.degree. C. is considered a high temperature but in noncentrifugal environments it is considered a low temperature. This is evidence that plastic applications in the ultracentrifuge impart a stress environment unusual for plastics and do not allow the use of material and design data generated in more conventional environments.
The liquid contents of a typical conical tube can exert more than 35,000 psi in the lower region of the adapter. This tremendous pressure in combination with self-induced load of the adapter and rotor bucket cause the bucket rotor to increase in diameter during centrifugation. The plastic adapter, captured between the highly pressurized fluid at the bottom of the horizontal spinning tube and the expanding internal surface of the rotor bucket, also increases in diameter. The rotor bucket having expanded within its elastic limit immediately returns to its original size after centrifugation, but the adapter and tube, constructed of polymers with visoelastic properties, decrease in diameter more slowly and are therefore tightly held in the rotor hole and adapter hole respectively for periods of 4-10 hours, overnight or longer, particularly after long multi-hour, high temperature centrifuging runs. It is the extremely high pressure and g-force fields and resulting difficulties in adapter/tube behavior that distinguish the application of the conical tube in ultracentrifuges from that of superspeed (i.e. 7000 rpm to 25,000 rpm) centrifuges.
Because clearances between the tube and adapter and adapter and rotor hole are significantly reduced after centrifugation, an adapter made from a material with a low coefficient of friction is desirable to facilitate removal of the tube from the adapter and the adapter from the rotor hole. Since pressures generated are so high, the outside tube surface is essentially hydroformed against the corresponding surface of the adapter. Any surface irregularities from machining or molding also can cause the tube to lock itself into the adapter.
Particularly in long hour runs, for example in concentrating RNA (ribonucleic acid), the use of Noryl plastic adapters of constant sidewell diameter resulted in severe sticking of both the sample tube in the rotor and the adapter in the rotor. Substituting of Delrin plastic adapters for the Noryl adapters to solve a simultaneous cracking problem failed to solve the sticking problem. Water was placed at the bottom of the rotor cavity to absorb the centrifuging stress and shock but this was not successful in solving the sticking problem.
Generally swinging bucket rotors can be operated at design speed with the buckets containing components of density less than 1.2 grams per cc. If any component, such as the tube, adapter material or fluid contents are greater than 1.2 grams per cc, the rotor speed has to be reduced so that the load on the rotor does not exceed design specifications. So that a variety of centrifuge tests can be performed, it is desirable that a tube adapter exhibits good chemical and stress cracking resistance and that it can be sterilized by various means such as a steam autoclaving or solvent immersion.
Although tube adapters have been previously developed for use in ultracentrifuge rotor bores, adapters have not been designed which could be used in swinging bucket rotors at maximum rated speed of the rotor and could be easily extracted from the rotor hole with no agitation of the contents of the tube. The swinging bucket rotor applies a symmetrical pressure and relative high g-field load to all components it contains. The use of tubes and adapters in vertical and fixed angle rotors has not been found to be as difficult since the centrifugal loading on the adapter is asymmetrical and applies a centrifugal vector component perpendicularly to the spin axis allowing a gap to exist between the adapter and hole on the inward or centripetal side of the adapter.